Quantification of α-hydroxy ceramides in mice serum by LC-MS/MS: Application to sepsis study

Alpha-hydroxy ceramides are not only the precursors of many complex sphingolipids, also play a major role in membrane homeostasis and cellular signal transduction. However, current research rarely involved quantitative methods for α-hydroxy ceramides, which greatly restricts the study of its biological function. This work aimed to develop a reliable assay for the accurate quantification of α-hydroxy ceramides in vivo study. LC-MS/MS method was developed for the accurate quantification of six α-hydroxy ceramides of Cer(d18:1/16:0(2OH)), Cer(d18:1/18:0(2OH)), Cer(d18:1/18:1(2OH)), Cer(d18:1/20:0(2OH)), Cer(d18:1/22:0(2OH)) and Cer(d18:1/24:1(2OH)) in mice serum. This assay was validated with low limit of quantitation of 3.125 ng/mL, a dynamic range of 3.125-400 ng/mL (R2 > 0.99), precision (<15 %), and accuracy (88 % to 115 %). Applying the method to the determination of α-hydroxy ceramides in the serum of sepsis mice, the levels of Cer(d18:1/16:0(2OH)), Cer(d18:1/20:0(2OH)), Cer(d18:1/24:1(2OH)) were significantly elevated in LPS-induced septic as compared to the normal control. In conclusion, this LC-MS method was qualified in α-hydroxy ceramides quantification in vivo and a significant association was found between α-hydroxy ceramides and sepsis.